There is societal need for new therapeutic agents in our arsenal of defenses against bacterial and fungal pathogens, many of which are increasingly resistant to existing antibiotics. Filamentous fungi are considered promising resources for the development of novel bioactive compounds because of their great potential to produce various kinds of secondary metabolites (SM), however, antibiotic discovery and production in fungi lags far behind bacteria. This research proposal advances sciences of fungal functional genomics to activate fungal silent SM clusters by using the newly developed fungal artificial chromosomes (FACs). Our purpose is to discover novel antibiotics and identify the best lead candidates for clinical development. Scientists at Intact Genomics Inc. and the University of Wisconsin at Madison will develop, utilize, and combine three aspects of novel technology innovation and genomic tools to enable therapeutic agent discovery in fungi. Specifically, the proposed research will identify antibiotic compounds using: 1) genetically enhanced A. nidulans strains, 2) in vitro BAC/FAC engineering, and 3) culture conditions with epigenetic modifications and bacterial co-culture. The primary objectives are to activate at least 5 of 40 silent and or cryptic SM gene clusters (FACs) of A. terreus for proof-of-concept using the above technologies and to screen these activated FACs against bacterial and fungal tester strains to discover novel antibacterial and antifungal properties. Our long-term goals are to develop a high through-put small molecule discovery platform in fungi in order to discover novel natural products from at least 1,000 silent SM pathways from completely sequenced fungal genomes. Moreover, we will characterize identified antimicrobial agents to determine the best lead candidates for clinical development. Lead candidates will have novel chemical structures, high potency against bacterial and or fungal pathogens, and minimal toxicity for eukaryotic cells. Each of the different technologies necessary for the proposed research has been proven effective separately; therefore, the combination of these different techniques has a high probability of success and also represents a significant advancement for the science of antibiotic discovery. In addition, the 1,000 activated silent SM clusters and their metabolites produced from this research are a valuable resource that may be screened for other bioactive compounds (e.g., with anticancer or antiviral activities) in subsequent research. 1